Methods for detecting and inhibiting angiogenesis using peptides

ABSTRACT

The present invention provides methods for reducing or inhibiting angiogenesis in a tissue, by contacting α5β1 integrin in the tissue with an agent that interferes with specific binding of the α5β1 integrin to a ligand expressed in the tissue; and methods of identifying angiogenesis in a tissue, by contacting the tissue with an agent that specifically binds α5β1 integrin, and detecting specific binding of the agent to β5β1 integrin associated with a blood vessel in the tissue. Also provided are methods of diagnosing a pathological condition characterized by angiogenesis in a tissue in an individual. The invention further provides methods of reducing or inhibiting angiogenesis in a tissue in an individual, by administering to the individual an agent that interferes with the specific binding of α5β1 integrin to a ligand expressed in the tissue; and methods of reducing the severity of a pathological condition associated with angiogenesis in an individual, by administering to the individual an agent that interferes with specific binding of α5β1 integrin to a ligand in a tissue associated with the pathological condition. The invention also provides methods of identifying an agent that reduces or inhibits angiogenesis associated with α5β1 integrin expression in a tissue by contacting a tissue exhibiting angiogenesis associated with α5β1 integrin expression with an agent, and detecting a reduction or inhibition of angiogenesis in the tissue.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of co-pending application Ser. No.10/685,665, filed Oct. 14, 2003, which is a continuation of applicationSer. No. 09/307,223, filed May 7, 1999, now U.S. Pat. No. 6,852,318,which claims the benefit of priority of U.S. Provisional ApplicationSer. No. 60/084,850 to Judith A. Varner, filed May 8, 1998, nowabandoned, and entitled A NOVEL METHOD FOR THE DETECTION AND INHIBITIONOF ANGIOGENESIS, the entire contents of which are incorporated herein byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made, in part, with government support under grantnumber R01 CA71619 awarded by the National Cancer Institute. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods for detecting and treatingconditions involving undesirable angiogenesis and more specifically tomethods of detecting or inhibiting angiogenesis by interfering withspecific binding of α5β1 integrin to a ligand.

2. Background of the Invention

Angiogenesis is the process whereby new blood vessels are formed.Angiogenesis, also called neovascularization, occurs normally duringembryogenesis and development, and occurs in fully developed organismsduring wound healing and placental development. In addition,angiogenesis occurs in various pathological conditions, including inocular diseases such as diabetic retinopathy and macular degenerationdue to neovascularization, in conditions associated with tissueinflammation such as rheumatoid arthritis and inflammatory boweldisease, and in cancer, where blood vessel formation in the growingtumor provides oxygen and nutrients to the tumor cells, as well asproviding a route via which tumor cells metastasize throughout the body.Since millions of people around the world are afflicted by thesediseases, a considerable effort has been made to understand themechanisms involved in angiogenesis in the hope that such anunderstanding will allow the development of methods for detecting andinhibiting such undesirable angiogenesis.

Angiogenesis occurs in response to stimulation by one or more knowngrowth factors, and also may involve other as yet unidentified factors.Endothelial cells, which are the cells that line mature blood vessels,normally do not proliferate. However, in response to an appropriatestimulus, the endothelial cells become activated and begin toproliferate and migrate into unvascularized tissue, to form new bloodvessels. In some cases, precursor cells can be activated todifferentiate into endothelial cells, which form new blood vessels.

Blood vessels are surrounded by an extracellular matrix. In addition tostimulation by growth factors, angiogenesis depends on interaction ofthe endothelial cells with the extracellular matrix, as well as witheach other. The activation of endothelial cells by growth factors andthe migration into and interaction with the extracellular matrix andwith each other is dependent on cell surface receptors expressed by theendothelial cells. These cell surface receptors, which include growthfactor receptors and integrins, interact specifically with particularmolecules.

In pathological conditions such as age-related macular degeneration anddiabetic retinopathy, decreasing availability of oxygen to the retinaresults in a hypoxic condition that stimulates the secretion ofangiogenic growth factors such as vascular endothelial growth factors(VEGF), which induce abnormal migration and proliferation of endothelialcells into tissues of the eye. Such vascularization in ocular tissuescan induce corneal scarring, retinal detachment and fluid accumulationin the choroid, each of which can adversely affect vision and lead toblindness.

Angiogenesis also is associated with the progression and exacerbation ofinflammatory diseases, including psoriasis, rheumatoid arthritis,osteoarthritis, and inflammatory bowel diseases such as ulcerativecolitis and Crohn's disease. In inflammatory arthritic disease, forexample, influx of lymphocytes into the region surrounding the jointsstimulates angiogenesis in the synovial lining. The increasedvasculature provides a means for greater influx of leukocytes, whichfacilitate the destruction of cartilage and bone in the joint.Angiogenic vascularization that occurs in inflammatory bowel diseaseresults in similar effects in the bowel.

The growth of capillaries into atherosclerotic plaques in the coronaryarteries represents another pathological condition associated withgrowth factor induced angiogenesis. Excessive blood flow intoneovascularized plaques can result in rupture and hemorrhage of theblood-filled plaques, releasing blood clots that can result in coronarythrombosis.

The involvement of angiogenesis in such diverse diseases as cancer,ocular disease and inflammatory diseases has led to an effort toidentify methods for specifically inhibiting angiogenesis as a means totreat these diseases. For cancer patients, such methods of treatment canprovide a substantial advantage over currently used methods such aschemotherapy, which kill or impair not only the target tumor cells, butalso normal cells in the patient, particularly proliferating normalcells such as blood cells, epithelial cells, and cells lining theintestinal lumen. Such non-specific killing by chemotherapeutic agentsresults in side effects that are, at best, unpleasant, and can oftenresult in unacceptable patient morbidity, or mortality. In fact, theundesirable side effects associated with cancer therapies often limitthe treatment a patient can receive.

For other pathological conditions associated with abnormal angiogenesissuch as diabetic retinopathy, there are no effective treatments short ofretinal transplants. However, even if retinal transplantation isperformed, the new retina would be subject to the same conditions thatresulted in the original retinopathy. Thus, there exists a need toidentify the molecular interactions involved in the undesirableangiogenesis that occurs in certain pathological conditions such thatmethods for diagnosing and specifically treating such pathologies can bedeveloped. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods for reducing or inhibitingangiogenesis in a tissue, by contacting α5β1 integrin associated withblood vessels in the tissue with an agent that interferes with specificbinding of the α5β1 integrin to a ligand expressed in the tissue,thereby reducing or inhibiting angiogenesis in the tissue. In oneembodiment, the agent is an α5β1 antagonist that does not substantiallyinterfere with the specific binding of an integrin other than α5β1integrin to its ligand, for example, αVβ3 integrin binding tovitronectin. In another embodiment, the α5β1 integrin ligand isfibronectin.

A method of the invention is useful, for example, for reducing orinhibiting angiogenesis in ocular tissue such as retina, macula orcornea; in skin; in synovial tissue; in intestinal tissue; or in bone.In addition, a method of the invention is useful for reducing orinhibiting angiogenesis in a neoplasm, which can be benign or malignantand, where malignant, can be a metastatic neoplasm. As such, theinvention provides medicaments, which contain α5β1 antagonists and areuseful for reducing or inhibiting angiogenesis in an individual. Anagent useful in practicing a method of the invention can be a peptide,for example, a peptide containing the amino acid sequence CRRETAWAC (SEQID NO: 1); an antibody, for example, an anti-α5β1 integrin antibody oran α5β1 integrin binding fragment thereof; or a nonpeptide, smallorganic molecule, for example, (S)-2-{(2,4,6-trimethylphenyl)sulfonyl}amino-3-{7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxy-2,7-diazaspiro-{4,4}-non-2en-3-yl}carbonylamino}propionic acid. An agent useful as an α5β1 antagonist can be linked to acytotoxin, for example, a cancer chemotherapeutic drug.

The invention also provides methods of identifying the presence ofangiogenesis in a tissue by contacting the tissue with an agent thatspecifically binds α5β1 integrin, and detecting specific binding of theagent to α5β1 integrin associated with a blood vessel in the tissue. Theagent can be a peptide, an antibody, or a nonpeptide, small organicmolecule, and can be linked to a detectable label, which can be detecteddirectly, or the presence of which can be detected due to itsinteraction with a particular reagent. Such a method is useful foridentifying the presence of angiogenesis in various tissues, includingin normal tissues such as embryonic tissue or placental tissue, ingranulation tissue, or in a tissue involved in a pathological conditionsuch as a neoplasm, a retinopathy, or an arthritic condition or otherinflammatory condition.

The invention further provides methods of diagnosing a pathologicalcondition characterized by angiogenesis in a tissue in an individual. Amethod of diagnosis can be performed, for example, by obtaining a sampleof the tissue from the individual, wherein, in an individual having thepathological condition, the tissue exhibits angiogenesis; contacting thesample with an agent that specifically binds α5β1 integrin; anddetecting specific binding of the agent to α5β1 integrin associated witha blood vessel in the tissue, thereby diagnosing a pathologicalcondition characterized by angiogenesis in the individual. Thepathological condition can involve the eye, for example, diabeticretinopathy or macular degeneration; the skin, for example, a hemangiomaor psoriasis; a joint, for example, rheumatoid arthritis orosteoarthritis; or the intestine, for example Crohn's disease orulcerative colitis; or can be a neoplasm, which can be benign ormalignant. A malignant neoplasm, which can be metastatic, can be, forexample, a breast carcinoma, colon carcinoma, ovarian carcinoma, orpancreatic carcinoma.

A method of diagnosing a pathological condition characterized byangiogenesis in a tissue in an individual also can be performed byadministering an agent that specifically binds α5β1 integrin to anindividual suspected of having the pathological condition; and detectingspecific binding of the agent to α5β1 integrin associated with a bloodvessel in the tissue. The agent can be detectably labeled, for example,by linking it to a moiety such as a radionuclide, a paramagneticmaterial or an X-ray attenuating material. The method of detecting canbe an in vivo imaging method such as a radionuclide imaging, positronemission tomography, computerized axial tomography, or magneticresonance imaging method, or can be an ex vivo method, wherein,following administration of the agent, a sample of the tissue isobtained from the individual, and specific binding of the agent in thesample is detected. Agent that is specifically bound to α5β1 integrin insuch a sample can be detected directly, for example, by detectingradioactivity due to the moiety linked to the agent, or can be detectedindirectly by contacting the specifically bound agent with a reagentthat specifically interacts with the agent, or with the moiety, anddetecting an interaction of the reagent with the agent or the moiety.

The present invention further provides methods of reducing or inhibitingangiogenesis in a tissue in an individual, by administering to theindividual an agent that interferes with the specific binding of α5β1integrin to a ligand expressed in the tissue, thereby reducing orinhibiting angiogenesis in the tissue in the individual. Also providedis a method of reducing the severity of a pathological conditionassociated with angiogenesis in an individual, by administering to theindividual an agent that interferes with specific binding of α5β1integrin to a ligand in a tissue associated with the pathologicalcondition, thereby reducing or inhibiting angiogenesis in the tissueand, consequently, reducing the severity of the pathological condition.The condition can be any pathological condition associated withangiogenesis, including a neoplasm, which can be a malignant neoplasm,for example, a carcinoma such as breast carcinoma, colon carcinoma,ovarian carcinoma or pancreatic carcinoma, or a sarcoma, mesothelioma,teratocarcinoma, an astrocytoma, glioblastoma, or other neoplasm,including a metastatic malignant neoplasm. The agent can be administeredby various routes, for example, intravenously, orally, or directly intothe region to be treated, for example, directly into a neoplastic tumor;via eye drops, where the pathological condition involves the eye; orintrasynovially, where the condition involves a joint.

The invention also provides methods of identifying an agent that reducesor inhibits angiogenesis associated with α5β1 integrin expression in atissue. Such a method, which is useful as a screening assay, can beperformed by contacting a tissue exhibiting angiogenesis associated withα5β1 integrin expression with an agent, and detecting a reduction orinhibition of angiogenesis in the tissue. Contacting of the tissue withthe agent can occur in vivo or ex vivo. Where the method is performedusing an in vitro format, it readily can be adapted for automated, highthroughput screening assays. The tissue can be any tissue that undergoesangiogenesis associated with α5β1 integrin expression, for example,malignant neoplastic tissue, and can be from any individual, including,for example, from a mammal, bird, reptile or amphibian.

BRIEF DESCRIPTION OF TEE DRAWINGS

FIG. 1 demonstrates the inhibitory effect of the nonpeptide smallorganic molecule, SJ749, on α5⁺ HT29 tumor cell adhesion to fibronectin.α5⁺ HT29 tumor cells were produced by transfecting HT29 cells with α5⁺cDNA.

FIG. 2 demonstrates the dose dependent inhibitory effect of SJ749 onblood vessel branch point formation in chorioallantoic membranes(CAM's). Angiogenesis was stimulated by treatment of the CAM's withbasic fibroblast growth factor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for detecting angiogenesis in atissue by identifying α5β1 binding to a ligand in a blood vessel in thetissue. Methods of diagnosing the presence of angiogenesis in anindividual also are provided. The invention further provides methods forreducing or inhibiting angiogenesis in a tissue by interfering with thespecific binding of α5β1 integrin toga ligand expressed in the tissue.Methods of reducing or inhibiting angiogenesis, which can be associatedwith a pathological condition, in an individual, also are provided.

Angiogenesis depends on the cooperation of various growth factors andcell adhesion events. The αV integrins have been shown to play criticalroles in angiogenesis, although studies using αV integrin null mice havesuggested that other adhesion receptors and their ligands also may beinvolved in angiogenesis. As disclosed herein, the integrin α5β1 and itsligand fibronectin are coordinately upregulated during growth factorstimulated angiogenesis and on blood vessels present in human tumorbiopsies, and the interaction of these molecules is required for theangiogenesis that occurs during and supports tumor growth in vivo, aswell as angiogenesis associated with various pathological conditions.

The development of vascular networks during embryogenesis or normal andpathological angiogenesis depends on stimulation induced by growthfactors (Breier and Risau, Trends in Cell Biology 6:454-456 (1996);Breier et al., Thromb. Haemost. 78:678-683 (1997); Folkman, Nature Med.1:27-31 (1995); Risau, Nature 386:671-674 (1997)) and on cellularinteractions with the extracellular matrix (Stromblad and Cheresh,Chemistry and Biology 3:881-885 (1996); Varner, Exs. 79:361-390 (1997);each of the publications cited in this disclosure is incorporated hereinby reference). Genetic and functional analyses indicate thatextracellular components and cell surface receptors regulate endothelialcell growth, survival and differentiation in vasculogenesis and inangiogenesis (George et al., Development 119:1079-1091 (1993); Yang etal., Development 119:1093-1105 (1993); Stromblad and Cheresh, supra,1996; Bloch et al., J. Cell Biol. 139: 265-278 (1997); Varner, supra,1997; Risau, supra, 1997; Bader et al., Cell 95:507-519 (1998)).

Blood vessels arise during embryogenesis by two processes,vasculogenesis and angiogenesis (Risau, supra, 1997), and the role ofgrowth factors in both processes is well established. For example,vascular endothelial growth factor (VEGF; Ferrara et al., Nature380:439-442 (1996)) and its receptors (de Vries et al., Science255:989-991 (1992); Fong et al., Nature 376:66-70 (1995); Millauer etal., Cell 72:835-846 (1993); Shalaby et al., Cell 89:981-990 (1997)),and basic fibroblast growth factor (bFGF; Basilico and Moscatelli, Adv.Cancer Res. 59:115-165 (1992)) promote the initial development of theembryonic vascular network, and are involved in the formation of newblood vessels from pre-existing vessels during development, woundhealing and the female reproductive cycle. VEGF (Warren et al., J. Clin.Invest. 95:1789-1797 (1995); Yoshida et al., Mol. Cell. Biol.17:14015-4023 (1997); Kong et al., Human Gene Ther. 9:823-833 (1998)),bFGF (Stan et al., J. Neurosurg. 82:1044-1052 (1995); Chopra et al., J.Canc. Res. Clin. Oncol. 123:167-172 (1997); Czubayko et al., Nature Med.3:1137-1140 (1997); Yoshida et. al., supra, 1997), Interleukin-8 (IL-8;Arenberg et al., J. Clin. Invest. 97: 2792-2802 (1996); Luca et al., Am.J. Path. 151:1105-1113 (1997); Keane et al., J. Immunol. 159:1437-43(1997); Yatsunami et al., Cancer Lett. 120:101-108 (1997); Yoshida etal., Invest. Ophthamol. Vis. Sci. 39:1097-1106 (1998)), and tumornecrosis factor-α (TNFα; Yoshida et. al., supra, 1997) are some of thegrowth factors that have a role in the angiogenesis that is associatedwith various pathological conditions, including, for example, solidtumor growth, diabetic retinopathy, and rheumatoid arthritis.

While growth factors stimulate new blood vessel growth, adhesion to theextracellular matrix (ECM) regulates endothelial cell survival,proliferation and motility during new blood vessel growth (Stromblad andCheresh, supra, 1996; Varner, supra, 1997). Specific integrins or theirligands also influence vascular development and angiogenesis. Forexample, the αV integrins participate in angiogenesis by providingsurvival signals to activated endothelial cells (Arap et al., Science279:377-380 (1997); Brooks et al., Science 264: 569-571 (1994a); Carronet al., Cancer Res. 58:1930-1955 (1998); Clark et al., Amer. J. Pathol.148:1407-1421 (1997); Drake et al., Devel. Dyn. 193:83-91 (1992); Clarket al., J. Cell Science 108:2655-2661 (1995); Friedlander et al.,Science 270:1500-1502 (1995)). However, some aspects of angiogenesisalso can proceed in the absence of αV integrins (Bader et. al., supra,1998), suggesting that other molecules, including the β1 integrinfamily, may compensate for the absence αV integrins during development(Drake et al., supra, 1992; Bloch et al., supra, 1997; Senger et al.,Proc. Natl. Acad. Sci., USA 94:13612-13617 (1997)).

While active roles for integrins in the promotion of angiogenesis havebeen identified, the cognate ECM ligands for integrins that are involvedin angiogenesis in vivo are less well described. One ECM protein,fibronectin, is expressed in provisional vascular matrices and providesproliferative signals to vascular cells during wound healing,atherosclerosis, and hypertension (Magnusson and Mosher, Arterioscler.Thromb. Vasc. Biol. 18:1363-1370 (1998)). Fibronectin expression isupregulated on blood vessels in granulation tissues during wound healing(Clark et al., J. Invest. Dermatol. 79:269-276 (1982)), and an isoformof fibronectin, the ED-B splice variant, is preferentially expressed onblood vessels in fetal and tumor tissues, but not on normal quiescentadult blood vessels (Castellani et al., Int. J. Cancer 59:612-618(1994); Kaczmarek et al., Int. J. Cancer 58:11-16 (1994); Neri et al.,Nature Biotech. 15:1271-1275 (1997)). These observations suggest thatfibronectin may have a role in angiogenesis. In addition, animals thatlack fibronectin die early in development from a collection of defects,including missing notochord and somites as well as an improperly formedvasculature (George et al., supra, 1993). Prior to the presentdisclosure, however, a direct functional role for fibronectin invasculogenesis or in angiogenesis was not established.

Several integrins bind to fibronectin (Hynes, Cell 69:11-25 (1992)), andintegrin α5β1 generally is selective for fibronectin (Pytela et al.,Cell 40:191-98 (1985)). Studies have demonstrated that loss of the geneencoding the integrin α5 subunit is embryonic lethal in mice and isassociated with a complete absence of the posterior somites and withsome vascular and cardiac defects (Yang et al., supra, 1993; Goh et al.,Development 124: 4309-4319 (1997)). It was unclear, however, whetherintegrin α5β1 has a direct role in the regulation of vasculardevelopment or of angiogenesis in particular.

As disclosed herein, both fibronectin and its receptor, α5β1 integrin,directly regulate angiogenesis. Moreover, the specific interaction offibronectin and α5β1 is central to the contribution of these twomolecules to angiogenesis. Integrin α5β1 participates in pathways ofangiogenesis that are the same as those of integrin αVβ3, but distinctfrom the pathways involving αVβ5. It is further disclosed herein thatagents that interfere with the specific binding of α5β1 and fibronectincan reduce or inhibit growth factor stimulated angiogenesis and theangiogenesis that occurs in tumors and, therefore, can be useful fortreating various pathological conditions, including malignant neoplasms.

The participation of the central cell binding domain of fibronectin andits receptor α5β1 in angiogenesis is disclosed herein. Expression ofboth integrin α5β1 and fibronectin were significantly enhanced on bloodvessels of human tumors and in growth factor stimulated tissues, whilethese molecules were minimally expressed on normal human vessels and onunstimulated tissues (Example I). In addition, antibody antagonists,which bind the central cell binding domain of fibronectin and anti-α5β1antibodies, as well as two other classes of α5β1 antagonists (peptidesand nonpeptide, small organic molecule antagonists) blocked growthfactor stimulated angiogenesis in chick chorioallantoic membrane (CAM;Example II) and in human skin grown on SCID mice (Example III).Antagonists of integrin α5β1 blocked bFGF, TNFα and IL-8 stimulatedangiogenesis, but had a minimal effect on VEGF-induced angiogenesis.Each of these α5β1 antagonists inhibited tumor angiogenesis and resultedin tumor regression in animal model systems (Example IV). Antagonists offibronectin function also blocked both bFGF and VEGF angiogenesis,suggesting that other fibronectin receptors are involved inVEGF-mediated angiogenesis.

The results disclosed herein demonstrate that the expression of integrinα5β1 and fibronectin in angiogenesis is coordinated. When the expressionof each molecule is minimal, as on unstimulated, quiescent bloodvessels, antagonists of each molecule and addition of fibronectin tochick chorioallantoic membranes (CAM's) had little effect onangiogenesis. In contrast, after stimulation with growth factors, α5β1and fibronectin expression are enhanced and blood vessels becomesensitive to agents that act as antagonists of either molecule, as wellas to the effects of exogenously added fibronectin. VEGF stimulationdoes not increase α5β1 expression, supporting the observation that VEGFangiogenesis is refractory to antagonists of α5β1. This result issubstantiated by a report that in vitro expression of integrin α5β1 onendothelial cells was upregulated in response to bFGF (Collo and Pepper,J. Cell Sci. 112:569-578 (1999)), and that VEGF failed to upregulateα5β1 expression (Senger et al., Am. J. Pathol. 149:1-7 (1996); Senger etal., Proc. Natl. Acad. Sci. USA 94:13612-13617 (1997)). Thus, thefunctional roles of integrin α5β1 and fibronectin in angiogenesis likelyare a direct consequence of their growth factor induced expression.

Antibodies directed against the central cell binding fragment offibronectin, which contains the RGD integrin binding site, inhibitedangiogenesis (Examples II and III). These antibodies likely interferewith the specific binding of α5β1 integrin to fibronectin, and,consequently, with possible downstream signal transduction events invivo. Stimulation of bFGF angiogenesis by fibronectin and its cellbinding domain in an α5β1-dependent manner indicate that α5β1 is theintegrin receptor for fibronectin during angiogenesis. The absence ofintegrin α5β1 expression in VEGF stimulated angiogenesis likely accountsfor the failure of fibronectin to enhance VEGF angiogenesis, even thoughantibodies directed against the cell binding peptide of fibronectinblocked VEGF angiogenesis. The results disclosed herein are the firstdemonstration of a direct in vivo role for fibronectin in angiogenesis.

The results disclosed herein also are the first to clearly identify arole for an extracellular matrix protein in the promotion ofangiogenesis. Although collagens have been suggested to have roles invascular development, intact collagens do not support endothelial celloutgrowth, survival or proliferation (Ilan et al., J. Cell Sci.111:3621-3631 (1998); Isik et al., J. Cell. Phys. 175:149-155 (1999)).In fact, inhibition of the collagen receptors integrins α2β1 and α1β1prevented the formation of large blood vessels and promoted theformation of small vessels (Senger et al., supra, 1997). Those resultssuggest that α2β1, α1β1, and their ligand, collagen, are involved inblood vessel maturation, rather than in the promotion of new bloodvessel sprouts.

A functional role for integrin α5β1 in angiogenesis was established bydemonstrating that agents that antagonize α5β1 binding to its ligandblocked angiogenesis induced by growth factors and angiogenesis in tumorfragments (Examples II, III and IV). Like α5β1, αVβ3 can serve as afibronectin receptor (Charo et al., J. Cell Biol. 111:2795-800 (1990)),although, as disclosed herein, endothelial cells use α5β1 as the majorfibronectin receptor when both integrins are expressed.

The expression of α5β1 and αVβ3 is regulated by similar growth factors,and both integrins have a significant role in bFGF, TNFα, IL-8 andtumor-induced angiogenesis, but not in VEGF-induced angiogenesis (seeExamples; see, also, Brooks et al., supra, 1994a; Brooks et al., Cell79:1157-1164 (1994b); Friedlander et al., supra, 1995). These twointegrins likely influence the same angiogenesis pathways, sincecombinations of their antagonists in angiogenesis animal models wereneither additive nor synergistic (see Example II).

Binding of integrins to extracelluar matrix proteins promotes cellattachment, migration, invasion, survival and proliferation (Varner,supra, 1997), and antagonists of αVβ3 induce apoptosis of proliferatingendothelial cells in vitro and in vivo (Brooks et al., supra, 1994b;Stromblad et al., supra, 1996). As disclosed herein, α5β1 antagonistsalso induce apoptosis of growth factor stimulated endothelial cells invitro and in vivo.

Antagonists of α5β1 blocked tumor angiogenesis and growth (Example IV),similar to antagonists of integrin αVβ3 (Brooks et al., supra, 1994b,1995). The tumor cell lines used for in vivo tumorigenicity andangiogenesis studies (Example IV) were integrin α5β1 negative, todiscount any direct effect of the antagonists on the tumor cells, andremained α5β1 negative through the course of their culture on CAM's.HT29 tumors express a variety of growth factors, including VEGF, TNFα,TGFα, TGFβ, PDGF and IL-8; it is not known whether HT29 cells alsoexpress bFGF. VEGF is most commonly associated with the hypoxic core ofthe tumor, and is transcriptionally regulated by hypoxia, whereas bFGFand other factors are associated with the growing edge of the tumor(Shweiki, et. al., supra, 1992; Kumar et al., Oncol. Res. 10:301-311(1998)). As observed for growth factor stimulated CAM's, α5β1antagonists did not impact large pre-existing vessels on the CAM thatunderlie the transplanted tumors. These results demonstrate that agentsthat interfere with specific binding of α5β1 to its ligands,particularly fibronectin, can reduce or inhibit angiogenesis. The use ofsuch agents, therefore, can provide a clinical benefit to individualssuffering from various pathological conditions, including to cancerpatients.

As used herein, the term “integrin” refers to the extracellularreceptors that are expressed in a wide variety of cells and bind tospecific ligands in the extracellular matrix. The specific ligands boundby integrins can contain an arginine-glycine-aspartic acid tripeptide(Arg-Gly-Asp; RGD) or a leucine-aspartic acid-valine tripeptide, andinclude, for example, fibronectin, vitronectin, osteopontin, tenascin,and von Willebrand's factor. The integrins comprise a superfamily ofheterodimers composed of an c subunit and a β subunit. Numerous αsubunits, designated, for example, αV, α5 and the like, and numerous βsubunits, designated, for example, β1, β2, β3, β5 and the like, havebeen identified, and various combinations of these subunits arerepresented in the integrin superfamily, including α5β1, αVβ3 and αVβ5.The superfamily of integrins can be subdivided into families, forexample, as αV-containing integrins, including αVβ3 and αVβ5, or theβ1-containing integrins, including α5β1 and αVβ1. Integrins areexpressed in a wide range of organisms, including C. elegans, Drosophilasp., amphibians, reptiles, birds, and mammals, including humans.

As disclosed herein, antibody, peptide and nonpeptide small organicmolecule antagonists of α5β1 can interfere with the specific binding ofα5β1 integrin with its ligands, particularly fibronectin, in vasculartissue, and can reduce or inhibit angiogenesis (see Examples II, III andIv). Such molecules that interfere with the specific binding of α5β1with its ligands are referred to herein generally as “agents,” “agentantagonists” or “α5β1 antagonists.” As used herein, the term “specificbinding” or “binds specifically,” when used in reference to theinteraction of two or more molecules, means that the molecules canassociate with each other under in vivo conditions and in vitro whenincubated under appropriate conditions, which can mimic in vivoconditions. The terms “specifically interact” and “specific association”also are used to refer to molecules that specifically bind.

For purposes of the present invention, the molecules that specificallyinteract with each other generally are a receptor-type molecule and itsligand, including, for example, an integrin and its particular ligand orligands, or an antibody and its particular antigen or antigens. It isrecognized, however, that other molecules, for example, an α integrinsubunit and a β integrin subunit also interact specifically to form anintegrin heterodimer, as can an α5β1 antagonist and an α5β1 integrin.Methods for determining whether two molecules specifically interact aredisclosed herein, and methods of determining binding affinity andspecificity are well known in the art (see, for example, Harlow andLane, Antibodies: A laboratory manual (Cold Spring Harbor LaboratoryPress, 1988); Friefelder, “Physical Biochemistry: Applications tobiochemistry and molecular biology” (W.H. Freeman and Co. 1976)).

Antibodies, peptides and nonpeptide small organic molecule antagoniststhat interfere with the specific binding of α5β1 with fibronectin areexemplified (see Example II). As used herein, the term “interfere,” whenused in reference to the action of an agent antagonist on the specificinteraction of a receptor and its ligand, means that the affinity of theinteraction is decreased below the level of binding that occurs in theabsence of the agent. The skilled artisan will recognize that theassociation of a receptor and its ligand is a dynamic relationship thatoccurs among a population of such molecules such that, at any particulartime, a certain proportion of receptors and ligands will be inassociation. An agent that interferes with the specific interaction of areceptor and its ligand, therefore, reduces the relative number of suchinteractions occurring at a given time and, in some cases, cancompletely inhibit all such associations.

The term “antagonist” is used herein to mean an agent, which can be anantibody, a peptide or a nonpeptide small organic molecule, that caninterfere with the specific interaction of a receptor and its ligand. Ananti-α5β1 integrin antibody, which can interfere with the binding ofα5β1 with fibronectin, thereby reducing or inhibiting the association ofα5β1 integrin with fibronectin, is an example of an α5β1 antagonist. Anantagonist can act as a competitive inhibitor or a noncompetitiveinhibitor of α5β1 binding to its ligand.

It can be difficult to distinguish whether an antagonist completelyinhibits the association of a receptor with its ligand or reduces theassociation below the limit of detection of a particular assay. Thus,the term “interfere” is used broadly herein to encompasses reducing orinhibiting the specific binding of a receptor and its ligand.Furthermore, an agent can interfere with the specific binding of areceptor and its ligand by various mechanism, including, for example, bybinding to the ligand binding site, thereby interfering with ligandbinding; by binding to a site other than the ligand binding site of thereceptor, but sterically interfering with ligand binding to thereceptor; by binding the receptor and causing a conformational or otherchange in the receptor, which interferes with binding of the ligand; orby other mechanisms. Similarly, the agent can bind to or otherwiseinteract with the ligand to interfere with its specifically interactingwith the receptor. For purposes of the methods disclosed herein forinterfering with the specific interaction of an α5β1 integrin and itsligand, an understanding of the mechanism by which the interferingoccurs is not required and no mechanism of action is proposed.

An agent that acts as an antagonist for α5β1 integrin binding to itsligand can be an antibody, particularly an anti-α5β1 antibody or ananti-fibronectin antibody. As used herein, the term “antibody” is usedin its broadest sense to include polyclonal and monoclonal antibodies,as well as antigen binding fragments of such antibodies. With regard toan anti-integrin antibody, particularly an anti-α5β1 antibody, the term“antigen” means an integrin, particularly an α5β1 integrin protein,polypeptide, or peptide portion thereof, which may or may not includesome or all of an RGD binding domain. An anti-α5β1 antibody, or antigenbinding fragment thereof, is characterized by having specific bindingactivity for an α5β1 integrin of at least about 1×10⁵ M⁻¹, generally atleast about 1×10⁶ M⁻¹, and particularly at least about 1×10⁷ M⁻¹. Fab,F(ab′)₂, Fd or Fv fragments of an anti-α5β1 antibody, which retainspecific binding activity for the α5β1 integrin are included within thedefinition of an antibody.

The term “antibody” as used herein encompasses naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known tothose skilled in the art (Winter and Harris, Immunol. Today 14:243-246(1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra,1988; Hilyard et al., Protein Engineering: A practical approach (IRLPress 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford UniversityPress 1995); each of which is incorporated herein by reference).

Anti-integrin antibodies, including anti-α5β1 antibodies, can bepurchased from a commercial source, for example, Chemicon, Inc.(Temecula Calif.), or can be raised using as an immunogen asubstantially purified full length integrin, which can be a humanintegrin, mouse integrin or other mammalian or nonmammalian integrinthat is prepared from natural sources or produced recombinantly, or apeptide portion of an integrin, which can include a portion of the RGDbinding domain, for example, a synthetic peptide. A non-immunogenicpeptide portion of an integrin such as a human α5β1 can be madeimmunogenic by coupling the hapten to a carrier molecule such bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressingthe peptide portion as a fusion protein. Various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art and described, for example, by Harlow and Lane (supra, 1988).

Particularly useful antibodies for performing a method of the inventionare those that specifically bind to an α5β1 integrin. Such antibodiesare particularly useful where they bind α5β1 with at least an order ofmagnitude greater affinity than they bind another integrin, for example,αVβ3 or αVβ5. An anti-fibronectin antibody also can be useful in amethod of the invention, particularly an anti-fibronectin antibody thatinterferes with binding of fibronectin to α5β1 integrin, but not to αVβ3or other integrins.

As disclosed herein, an anti-α5β1 antibody was used to detect regions ofgrowth factor stimulated angiogenesis, as occurs in a pathologicalcondition (see Example I). The presence or amount of α5β1 integrinexpression can be identified, for example, in a tissue sample, which canbe a histological section obtained from a tissue or organ of anindividual suspected of having a pathology characterized, at least inpart, by undesirable angiogenesis. The identification of the presence orlevel of an α5β1 integrin expression in the sample can be made usingwell known immunoassay or immunohistochemical methods (Harlow and Lane,supra, 1988). An anti-α5β1 antibody, particularly an antibody thatprevents ligand binding to the α5β1 integrin, also can be used in ascreening assay to identify agents that compete for ligand binding tothe integrin. As disclosed herein, such agents can be useful forinhibiting α5β1 mediated angiogenesis.

Peptides that specifically bind to α5β1 also are useful as antagonistsof α5β1 binding to its ligands, including fibronectin. As discussed foranti-α5β1 antibodies, a peptide that specifically binds α5β1 can beuseful in a method of the invention where the antibody binds to α5β1with at least about a two-fold greater specificity than it binds toanother integrin, for example, αVβ3, is more useful if it has at leastabout a five-fold greater specificity for α5β1, and is particularlyuseful if it has at least about a one order of magnitude greaterspecificity for α5β1 than for an integrin such as αVβ3. As such, thevarious RGD and RLD containing peptides that have been identified basedon their relatively high binding affinity for αVβ3 or for αVβ5(PCT/US94/13542) are not considered peptide antagonists of α5β1 bindingto its ligand, as defined herein.

The term “peptide” is used broadly herein to include oligomers andpolymers of amino acids or amino acid analogs that are linked by apeptide bond or an analog of a peptide bond. As such, the term “peptide”includes molecules commonly referred to as peptides, which generallycontain about two to about fifty amino acids, as polypeptides, whichgenerally contain about twenty to fifty amino acids or more, and asproteins, which can include peptides or polypeptides that, for example,are post-translationally modified. Thus, peptide antagonists contain twoor more amino acids, which can be L-amino acids or D-amino acids,chemically modified amino acids, which can be naturally occurring ornon-naturally occurring amino acids, or amino acid analogs. Peptidesuseful as α5β1 antagonists that reduce or inhibit angiogenesis can beidentified by screening libraries of peptides, which can be preparedusing well known methods of chemical synthesis (see, for example,Koivunen et al., supra 1993, 1994), or can be purchased from commercialsources.

An agent that interferes with α5β1 binding to its ligand also can be anonpeptide, small organic molecule, including a peptidomimetic, which isan organic molecules that mimics the structure of a peptide; or apeptoid such as a vinylogous peptoid. A nonpeptide small organicmolecule that acts as an antagonist to the specific interaction of α5β1integrin binding to a ligand, fibronectin, can be, for example, aheterocycle having the general structure(S)-2-phenylsulfonylamino-3-{{{8-(2-pyridinylaminomethyl)-}-1-oxa-2-azaspiro-{4,5}-dec-2-en-yl}carbonylamino}propionic acid, as exemplified herein by the molecule designated SJ749,which has the structure: (S)-2-{(2,4,6-trimethylphenyl)sulfonyl}amino-3-{7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-{4,4}-non-2-en-3-yl}carbonylamino}propionic acid (see Examples II and IV; U.S. Pat. No. 5,760,029). Asdisclosed herein, SJ749 interfered with α5β1 binding to fibronectin andreduced or inhibited angiogenesis in a dose dependent manner (see FIG.2). Additional nonpeptide, small organic molecule α5β1 antagonistsuseful in a method of the invention can be identified by screening, forexample, chemically modified derivatives of a heterocycle having thestructure disclosed above, including chemically modified derivatives ofSJ749, or other libraries of nonpeptide, small organic molecules (seebelow).

The present invention provides methods for reducing or inhibitingangiogenesis in a tissue, by contacting α5β1 integrin in the tissue withan agent that interferes with specific binding of the α5β1 integrin to aligand expressed in the tissue, thereby reducing or inhibitingangiogenesis in the tissue. A particularly useful agent antagonistinterferes with the binding of α5β1 to fibronectin, but does notsubstantially interfere with the specific binding of the ligand to anintegrin other than α5β1 integrin. As disclosed herein, an agent such asan anti-α5β1 antibody, a peptide, or a nonpeptide small organic moleculethat interferes with binding of α5β1 integrin to its ligand can reduceor inhibit growth factor stimulated angiogenesis and angiogenesis thatoccurs during tumor growth (see Examples II, III and IV).

As used herein, the phrase “reduce or inhibit,” when used in referenceto angiogenesis, means that the amount of new blood vessel formationthat occurs in the presence of an agent antagonist is decreased belowthe amount of blood vessel formation that occurs in the absence of anexogenously added agent antagonist. The terms “reduce” and “inhibit” areused together because it is recognized that the amount of angiogenesiscan be decreased below a level detectable by a particular assay methodand, therefore, it may not be possible to determine whether angiogenesisis reduced to a very low level or completely inhibited. Nevertheless, itwill be clear from the particular assay being used that, in response toan agent that interferes with α5β1 integrin to its ligand, angiogenesisin a tissue is decreased below the level of angiogenesis incorresponding untreated tissue. Methods for determining an amount ofblood vessel formation in a tissue, including the immunohistochemicalmethods disclosed herein (Example I), are well known in the art.

A method of the invention is useful, for example, for reducing orinhibiting angiogenesis in ocular tissue such as retina, macula orcornea; in skin such as occurs with psoriasis; in synovial tissue; inbone; or in intestinal tissue, by interfering with α5β1 binding to aligand such as fibronectin in the tissue. In addition, a method of theinvention is useful for reducing or inhibiting angiogenesis in aneoplasm, which can be benign or malignant and, where malignant, can bea metastatic neoplasm. An agent useful in practicing a method of theinvention can be a peptide, for example, a peptide containing the aminoacid sequence CRRETAWAC (SEQ ID NO: 1); an antibody, for example, ananti-α5β1 integrin antibody or an α5β1 integrin binding fragmentthereof; or a nonpeptide, small organic molecule, for example,(S)-2-{(2,4,6-trimethylphenyl)sulfonyl}amino-3-{7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxy-2,7-diazaspiro-{4,4}-non-2-en-3-yl}carbonylamino}propionic acid (SJ749). If desired, the agent can be linked to acytotoxin such as ricin or a cancer chemotherapeutic drug, providedlinkage of the cytotoxin does not substantially reduce the ability ofthe agent to specifically bind α5β1 integrin and interfere with thebinding of α5β1 to its ligand.

The invention also provides methods of identifying the presence ofangiogenesis in a tissue, by contacting the tissue with an agent thatspecifically binds α5β1 integrin, and detecting specific binding of theagent to α5β1 integrin associated with a blood vessel in the tissue,thereby identifying the presence of angiogenesis in the tissue. Theagent can be a peptide, an antibody, or a nonpeptide, small organicmolecule, and can be linked to a detectable label, which can be detecteddirectly, or the presence or which can be detected due to itsinteraction with a particular reagent. Such a method is useful foridentifying the presence of angiogenesis in various tissues, including,for example, normal tissues such as embryonic tissue or placentaltissue, granulation tissue, and a tissue involved in a pathologicalcondition. As such, the invention further provides methods of diagnosinga pathological condition characterized by angiogenesis associated withα5β1 integrin expression in a tissue in an individual.

The term “pathological condition” is used broadly herein to mean anyabnormal physical or physiological condition characterized, at least inpart, by angiogenesis associated with α5β1 integrin expression on newlyforming blood vessels in a tissue. Such pathological conditions areexemplified by neoplasms (see Example I), ocular diseases such asdiabetic retinopathy and macular degeneration associated withneovascularization, skin diseases such as psoriasis and hemangiomas,gingivitis, arthritic conditions such as rheumatoid arthritis andosteoarthritis, and inflammatory bowel diseases. Other pathologicalconditions amenable to a diagnostic or other method of the invention canbe identified using methods such as those disclosed in Example I orotherwise known in the art.

The term “neoplasm” is used broadly herein to mean any new, pathologicaltissue growth. For purposes of the present invention, a neoplasmgenerally results in the formation of a tumor, which is characterized,in part, by angiogenesis. A neoplasm can be benign, for example, ahemangioma, glioma, teratoma, and the like, or can be malignant, forexample, a carcinoma, sarcoma, glioblastoma, astrocytoma, neuroblastoma,retinoblastoma, and the like. The term “tumor” is used generally torefer to a benign or malignant neoplasm, and the term “cancer” is usedgenerally to refer to a malignant neoplasm, which may or may not bemetastatic. Malignant neoplasms that can be diagnosed using a method ofthe invention include, for example, carcinomas such as lung cancer,breast cancer, prostate cancer, cervical cancer, pancreatic cancer,colon cancer and ovarian cancer; and sarcomas such as osteosarcoma andKaposi's sarcoma, provided the neoplasm is characterized, at least inpart, by angiogenesis associated with α5β1 expression by the newlyforming blood vessels (see Examples I and III).

A method of diagnosis can be performed, for example, by obtaining asample of the tissue from the individual, wherein, in an individualhaving the pathological condition, the tissue exhibits angiogenesis;contacting the sample with an agent that specifically binds α5β1integrin; and detecting specific binding of the agent to α5β1 integrinassociated with a blood vessel in the tissue. An individual to bediagnosed or treated using a method of the invention can be anyindividual exhibiting angiogenesis associated with α5β1 integrinexpression and, therefore, can be, for example, a vertebrate such as amammal, including a human, dog, cat, horse, cow, or goat; a bird; or anyother animal, particularly a commercially important animal or adomesticated animal.

A method of diagnosing a pathological condition characterized byangiogenesis in a tissue in an individual also can be performed byadministering an agent that specifically binds α5β1 integrin to anindividual suspected of having the pathological condition; and detectingspecific binding of the agent to α5β1 integrin associated with a bloodvessel in the tissue. The agent can be detectably labeled, for example,by linking the agent to a moiety, which is selected based, for example,on whether specific binding of the agent is to be detected in vivo orwhether a tissue to which the agent is suspected of binding is to beremoved, for example, by biopsy, and examined ex vivo.

A moiety useful for labeling an agent antagonist can be a radionuclide,a paramagnetic material, an X-ray attenuating material, a fluorescent,chemiluminescent or luminescent molecule, a molecule such as biotin, ora molecule that can be visualized upon reaction with a particularreagent, for example, a substrate for an enzyme or an epitope for anantibody. The moiety can be linked to an agent using well known methods,which are selected, in part, based on the chemical nature of the agentand the moiety. For example, where the moiety is an amino acid sequencesuch as a hexahistidine (His6) sequence, and the agent is a peptide, theHis6 sequence can be synthesized as part of the peptide, and theHis6-labeled agent can be identified by the binding of a nickel ionreagent to the His6 moiety. Methods for chemically linking a moiety toan agent also can be utilized (see, for example, Hermanson, BioconjugateTechniques, (Academic Press 1996), which is incorporated herein byreference).

A specifically bound agent can be detected in an individual using an invivo imaging method such as a radionuclide imaging, positron emissiontomography, computerized axial tomography, or magnetic resonance imagingmethod, or can be detected using an ex viva method, wherein, followingadministration, a sample of the tissue is obtained from the individual,and specific binding of the agent in the sample is detected. An agentthat is specifically bound to α5β1 integrin in a sample can be detecteddirectly, for example, by detecting the agent or by detecting thepresence of a moiety such as by detecting radioactivity emitted by aradionuclide moiety. Specifically bound agent also can be detectedindirectly by further contacting it with a reagent that specificallyinteracts with the agent, or with a moiety linked to the agent, anddetecting interaction of the reagent with the agent or label. Forexample, the moiety can be detected by contacting it with an antibodythat specifically binds the moiety, particularly when the moiety islinked to the agent. The moiety also can be, for example, a substrate,which is contacted by an enzyme that interacts with and changes themoiety such that its presence can be detected. Such indirect detectionsystems, which include the use of enzymes such as alkaline phosphatase,horseradish peroxidase, beta-galactosidase and the like, are well knownin the art and commercially available, as are the methods forincorporating or linking the particular moiety to a particular type ofagent.

The present invention further provides methods of reducing or inhibitingangiogenesis in a tissue in an individual, by administering to theindividual an agent that interferes with the specific binding of α5β1integrin to a ligand expressed in the tissue, thereby reducing orinhibiting angiogenesis in the tissue in the individual. As such, theinvention provides methods of reducing the severity of a pathologicalcondition associated with angiogenesis in an individual, byadministering to the individual an agent that interferes with specificbinding of α5β1 integrin to a ligand in a tissue associated with thepathological condition, thereby reducing or inhibiting angiogenesis inthe tissue, and, consequently, reducing the severity of the pathologicalcondition.

As used herein, the term “reducing the severity of a pathologicalcondition” means that adverse clinical signs or symptoms associated withthe pathological condition are ameliorated. A reduction in the severityof a pathologic condition can be detected by various methods, includingroutine clinical tests such as blood tests, which can used to determinerelevant enzyme levels or circulating antigen or antibody; imagingtests, which can be used to detect a decrease in the growth rate or sizeof a neoplasm; or an ophthalmic procedure, which can be used to identifya reduction in the number of blood vessels in the retina of a diabeticpatient. Such clinical tests are selected based on the particularpathological condition being treated. A reduction in the severity of apathological condition also can be detected based on comments made bythe patient being treated, for example, that a patient suffering fromarthritis feels less pain or has greater joint mobility, or that apatient with diabetic retinopathy or with macular degeneration due toneovascularization can see more clearly, or the like.

Where an agent that interferes with the specific binding of an α5β1integrin to its ligand is to be administered to a living individual, forexample, for a diagnostic or therapeutic procedure, the agent generallywill be in the form of a pharmaceutical compositions comprising theagent or agents and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known in the art andinclude aqueous solutions such as physiologically buffered saline orother buffers or solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters. The selection of apharmaceutically acceptable carrier will depend, in part, on thechemical nature of the agent, for example, whether the agent is anantibody, a peptide or a nonpeptide, small organic molecule.

A pharmaceutically acceptable carrier can physiologically acceptablecompounds that act, for example, to stabilize the agent or increase itsabsorption, or other excipients as desired. Physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the agent and on the particular physio-chemicalcharacteristics of the agent.

Angiogenesis associated with α5β1 integrin expression can occur locally,for example, in the retina of an individual suffering from diabeticretinopathy, or can occur more systemically, for example, in anindividual suffering from rheumatoid arthritis or a metastatic malignantneoplasm. Since regions of such angiogenesis can be localized or canmore systemically dispersed, one skilled in the art would select aparticular route and method of administration of an agent thatinterferes with the specific binding of an α5β1 integrin with itsligand, for example, fibronectin, based, in part, on this factor. Forexample, in an individual suffering from diabetic retinopathy, whereangiogenesis associated with α5β1 integrin expression is localized tothe retina, the agent can be formulated in a pharmaceutical compositionconvenient for use as eye drops, which can be administered directly tothe eye. In comparison, in an individual suffering from a metastaticcarcinoma, the agent in a pharmaceutical composition that can beadministered intravenously, orally or by another method that distributesthe agent systemically. Thus, an agent antagonist can be administered byvarious routes, for example, intravenously, orally, or directly into theregion to be treated, for example, directly into a neoplastic tumor; viaeye drops, where the pathological condition involves the eye; orintrasynovially, where the condition involves a joint.

The amount of an α5β1 agent antagonist that is administered to anindividual will depend, in part, on whether the agent is administeredfor a diagnostic purpose or for a therapeutic purpose. Methods fordetermining an effective amount of an agent to administer for adiagnostic or a therapeutic procedure are well known in the art andinclude phase I, phase II and phase III clinical trials. An agent isadministered in an effective amount, which is an amount sufficient tointerfere with the specific binding of α5β1 integrin to its specificligand in an individual. Generally, an agent antagonist is administeredin a dose of about 0.0001 to 100 mg/kg body weight.

As disclosed herein, systemic administration of 5 μg anti-α5β1antibody/2 ml blood volume of chick embryo inhibited 50% of the growthfactor stimulated angiogenesis (Example II). Similarly, administrationof 120 picomoles of CRRETAWAC (SEQ ID NO: 1)/2 ml blood volume, andadministration of 15 picomoles of SJ749/2 ml blood volume inhibitedangiogenesis by 50%. Based on these results, the skilled artisan canestimate the amounts of such agents required to effectively inhibitangiogenesis in a tissue in an individual such as a human, and routineclinical trials can be used to determine optimal dosages. Assuming, forexample, that a human has a blood volume of about six liters, theartisan would know that a range of amounts less than or around about 15milligrams of an anti-α5β1 antibody can be used in a clinical trial fordetermining an amount of the agent to be administered to a human.Estimates of an amount to be administered can be adjusted accordingly,for example, where the agent is to be administered locally.

The total amount of an agent antagonist can be administered to a subjectas a single dose, either as a bolus or by infusion over a relativelyshort period of time, or can be administered using a fractionatedtreatment protocol, in which the multiple doses are administered over amore prolonged period of time. One skilled in the art would know thatthe concentration of a particular agent required to provide an effectiveamount to a region or regions of angiogenesis associated with α5β1integrin expression in an individual depends on many factors includingthe age and general health of the subject as well as the route ofadministration, the number of treatments to be administered, and thenature of the agent, including whether the agent is an antibody, apeptide, or a non-peptide small organic molecule. In view of thesefactors, the skilled artisan would adjust the particular dose so as toobtain an effective amount for efficaciously interfering with thespecific binding of α5β1 integrin with its ligand, thereby allowingeither for detection of the agent at a region of angiogenesis associatedwith α5β1 integrin expression for diagnostic purposes, or for reducingor inhibiting such angiogenesis for therapeutic purposes.

An agent useful for detecting or reducing or inhibiting angiogenesisassociated with α5β1 integrin expression, or a pharmaceuticalcomposition thereof containing the agent, can be used for treating anypathological condition that is characterized, at least in part, by suchangiogenesis. One skilled in the art would know that the agent can beadministered by various routes including, for example, orally, orparenterally, including intravenously, intramuscularly, subcutaneously,intraorbitally, intracapsularly, intrasynovially, intraperitoneally,intracisternally or by passive or facilitated absorption through theskin using, for example, a skin patch or transdermal iontophoresis.Furthermore, the agent can be administered by injection, intubation, viaa suppository, orally or topically, the latter of which can be passive,for example, by direct application of an ointment or powder containingthe agent, or active, for example, using a nasal spray or inhalant. Theagent can also be administered as a topical spray, if desire, in whichcase one component of the composition is an appropriate propellant. Thepharmaceutical composition also can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices (Gregoriadis, LiposomeTechnology, Vol. 1 (CRC Press, Boca Raton, Fla. 1984), which isincorporated herein by reference). Liposomes, for example, which consistof phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer.

As disclosed herein, agents that interfere with α5β1 integrin binding toits ligand can reduce or inhibit angiogenesis associated with α5β1expression. In addition to the exemplified agent antagonists, other suchagents can be identified by detecting agents that interfere α5β1integrin binding to its ligand. Thus, the invention provides screeningassays, which are useful for identifying an agent that reduces orinhibits angiogenesis associated with α5β1 integrin expression in atissue.

A screening assay of the invention can be performed by contacting atissue exhibiting angiogenesis associated with α5β1 integrin expressionwith an agent, and detecting a reduction or inhibition of angiogenesisin the tissue, thereby identifying an agent that reduces or inhibitsangiogenesis associated with α5β1 integrin expression in a tissue. Atissue can be contacted with the agent in vivo or ex vivo (see, forexample, U.S. Pat. No. 5,622,699). Where a screening method of theinvention is performed using an in vitro format, the can be adapted toautomated procedure, thus allowing high throughput screening assays forexamining libraries of molecules to identify potential α5β1 antagonists,which can reduce or inhibit angiogenesis associated with α5β1expression. The tissue can be any tissue that undergoes angiogenesisassociated with α5β1 integrin expression, for example, malignantneoplastic tissue.

Methods for preparing libraries of molecules, which can be screenedusing a method of the invention to identify α5β1 antagonists, whichreduce or inhibit angiogenesis associated with α5β1 expression,including, for example, oligonucleotide libraries (Gold et al., U.S.Pat. No. 5,270,163); peptide libraries (Koivunen et al., supra, 1993,1994); peptidomimetic libraries (Blondelle et al., Trends Anal. Chem.,14:83-92 (1995)); oligosaccharide libraries (York et al., Carb. Res.,285:99-128, (1996); Liang et al., Science, 274:1520-1522, (1996); andDing et al., Adv. Expt. Med. Biol., 376:261-269, (1995)); lipoproteinlibraries (de Kruif et al., FEBS Lett., 399:232-236, (1996));glycoprotein or glycolipid libraries (Karaoglu et al., J. Cell Biol.,130:567-577 (1995)); or chemical libraries containing, for example,drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem.,37:1385-1401 (1994); Ecker and Crook, Bio/Technoloqy, 13:351-360(1995)), including, for example, heterocycles having the generalstructure (S)-2-phenylsulfonylamino-3-{{{8-(2-pyridinylaminomethyl)-}-1-oxa-2-azaspiro-{4,5}-dec-2-en-yl}carbonylamino}propionic acid (U.S. Pat. No. 5,760,029). Libraries of diverse moleculesalso can be obtained from commercial sources.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I α5β1 Integrin is Expressed During Angiogenesis

This Example provides immunohistochemical evidence that α5β1 isexpressed in association with newly formed blood vessels in varioushuman and mouse tumors.

Five μm frozen sections of human normal breast and colon, coloncarcinoma, breast carcinoma, human tumor xenotransplants in six week oldCB17 female SCID mice (Charles River; Wilmington Mass.), and in breasttumors from Mtag mice were fixed for 1 min in acetone, air dried andrehydrated for 5 min in phosphate buffered saline (PBS). Sections wereblocked for 2 hr in 8% normal goat serum in PBS and incubated with: 1) 5μg/ml anti-α5β1 cytoplasmic tail polyclonal antibody (AB1928P;Pharmingen, Inc.; San Diego Calif.) and 5 μg/ml murine anti-human CD31monoclonal antibody (PECAM; MA-3100; Endogen); 2) 5 μg/ml anti-α5β1monoclonal antibody and 5 μg/ml rabbit anti-von Willebrand's factorantibody (016P; Biogenex; San Ramon Calif.); or 3) 5 μg/mlanti-fibronectin cell binding peptide monoclonal antibody (784A2A6;Chemicon, Inc.; Temecula Calif.) and 5 μg/ml anti-von Willebrand'sfactor antibody (016P), in 20 bovine serum albumin (BSA) in PBS for 2 hrat room temperature (RT).

Sections were washed by dipping in six fresh changes of PBS andincubated in 1:400-1:600 dilutions of goat anti-rabbit-FITC and in1:400-1:600 goat anti-mouse-rhodamine for 1 hr at RT (cross-absorbedsecondary antibodies; Biosource International; Camarillo Calif.). Slideswere washed, and coverslips were mounted in one drop of Fluoromount-G(Southern Biotechnology Associates; Birmingham Ala.) prior to digitalimage analysis under fluorescent illumination using a supercooled CCDcamera.

Analysis of frozen sections of human colon carcinoma and breastcarcinoma for expression of the endothelial cell marker CD31 (PECAM) andintegrin α5β1 by two color immunohistochemistry indicated that CD31positive tumor vessels (stained red) also were positive for integrinα5β1 expression (stained green); vessels positive for both moleculesappeared yellow by photomicrography. Large vessels with lumens, as wellas large and small vessels without apparent lumens stained positivelyfor integrin α5β1 and CD31. Sections of ovarian and pancreatic carcinomashowed similar patterns of integrin α5β1 expression on blood vessels. Incontrast, CD31 positive blood vessels present in sections of normalhuman colon and breast were negative for integrin α5β1, as were bloodvessels in other normal adult tissues, including skin. These resultsdemonstrate that integrin α5β1 expression is upregulated on tumorvasculature and that the majority of blood vessels in these tumorsections are positive for α5β1 expression. The results furtherdemonstrate that α5β1 is not significantly expressed on blood vessels innormal adult tissues.

Tumor tissues also were stained with antibodies directed againstfibronectin (stained red) and von Willebrand's Factor (stained green),which is another blood vessel marker. Examination of frozen sections ofbreast carcinoma and colon carcinoma, as well as normal human breast andcolon indicated that the extracellular matrix surrounding tumor vesselswas positive for fibronectin expression. In contrast, blood vessels innormal tissues expressed little, if any, fibronectin. Sections ofovarian and pancreatic carcinoma showed similar patterns of fibronectinexpression on blood vessels.

Notably, the expression of integrin α5β1 and its ligand, fibronectin,were coordinately upregulated on many of the same blood vessels withinhuman tumor sections. These coordinate expression of these molecules inhuman tumor tissues is indicative of a possible functional interactionbetween these proteins. Expression of integrin α5β1 and fibronectin alsowere observed on tumor vasculature in animal models of neoplasia,including human M21L melanoma tumor xenotransplants in SCID mice andspontaneous mammary tumors in the PyV mouse (see Guy et al., Mol. Cell.Biol. 12:954-961 (1992), which is incorporated herein by reference,regarding PyV mouse model). Thus, significantly elevated expression ofintegrin α5β1 and fibronectin is associated with the vasculature inspontaneous tumors and in experimentally induced human and murine tumorscompared to normal tissues.

EXAMPLE II α5β1 and Fibronectin are Required for Angiogenesis

This example demonstrates that fibronectin and the fibronectin receptorintegrin α5β1 are involved in angiogenesis in tumors and in growthfactor stimulated angiogenesis.

A. Methods

1. Cell Adhesion Assay

HT29 integrin α5β1⁺ cells, integrin α5β1⁻ colon carcinoma cells (Varneret al., Mol. Biol. Cell 6:725-740 (1995)), and chick embryo fibroblasts(CEF's) were maintained in DMEM high glucose supplemented with 10% fetalbovine serum (FBS) and gentamycin. Human umbilical vein endothelialcells (HUVEC's) were maintained in M199 medium containing sodiumbicarbonate, HEPES, heparin, endothelial cell growth supplement, 20% FBSand gentamycin. Culture media and reagents were from Irvine Scientific(Irvine, Calif.).

The wells of 48 well culture dishes (Costar, Inc.) were coated with 1μg/ml vitronectin, 2 μg/ml fibronectin (chick embryo fibroblasts andHUVEC's) or 10 μg/ml fibronectin (HT29-α5⁺ cells) for 1 hr at 37° C.,then blocked with 2% heat denatured BSA in PBS for 1 hr. Fifty thousandcells in 250 μl of adhesion buffer were added to triplicate wellscontaining 250 μl of a solution of 50 μg/ml of an anti-α5β1 functionblocking antibody (NKI-SAM-1, JBS5 or IIA1), 50 μg/ml of an anti-α5β1non-function blocking antibody (HA5 or VC5; Pharmingen, Inc.; San DiegoCalif.), 10 μM cyclic peptides (Koivunen et al., J. Biol. Chem.268:20205-20210 (1993); Koivunen et al., J. Cell Biol. 124:373380(1994)), 0-10 μM SJ749((S)-2-{(2,4,6-trimethylphenyl)sulfonyl}amino-3-{7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-{4,4}-non-2-en-3-yl}carbonylamino)propionic acid)), 50 μg/ml of LM609, an anti-αVβ3 function blockingantibody, 50 μg/ml P4C10, an anti-β1 function blocking antibody, 50μg/ml of an anti-fibronectin cell binding domain monoclonal antibody or50 μg/ml of an anti-fibronectin N-terminus monoclonal antibody inadhesion buffer (HEPES buffered Hanks balanced salt solution, HBSS,containing 1% BSA, 2 mM MgCl₂, 2 mM CaCl₂ and 0.2 mM MnCl₂).

Cells were allowed to adhere to dishes for 20 min at 37° C. Nonadherentcells were removed by washing each well four times with 500 μl of warmadhesion buffer. Adherent cells were then fixed for 15 min with 3.7%paraformaldehyde in PBS and stained with a 2% crystal violet solution.After extensive water washing to remove excess crystal violet, plateswere dried overnight. Crystal violet was extracted by incubation for 15min in 10% acetic acid and absorbance at 562 nm determined as anindicator of number of cells bound. Each experiment was performed intriplicate, with triplicate samples per condition. Data was presented aspercent of adhesion exhibited by the positive control (adhesion mediumalone)+/−standard error of measurement.

2. Cell Migration Assays

The lower side of 8 μm pore transwell inserts (Costar, Inc.) were coatedwith 2 μg/ml of fibronectin, collagen (Collaborative BiomedicalProducts; Bedford Mass.) or no protein for 1 hr and were blocked with 2%BSA in PBS for 1 hr. The inserts then were placed into 24 well culturedishes containing 500 μl migration buffer in the lower chamber.Twenty-five thousand HUVEC's in 50 μl of migration buffer (HEPESbuffered M199 medium containing 1% BSA, 2 mM MgCl₂, 2 mM CaCl₂ and 0.2mM MnCl₂) were added to the upper chamber of duplicate insertscontaining 50 μl of a solution of 50 μg/ml of anti-α5β1 functionblocking antibody (NKI-SAM-1, JBS5 or IIA1), 50 μg/ml of anti-α5β1non-function blocking antibody (HA5 or VC5), or 50 μg/ml of LM609 (ananti-αVβ3 function blocking antibody) in migration buffer, or migrationbuffer alone.

Cells were allowed to migrate from the upper to the lower chamber for 4hr at 37° C. Nonmigratory cells were removed from the upper surface bywiping the upper side with an absorbant tip, and cells that had migratedto the lower side of the transwell insert were fixed for 15 min with3.7% paraformaldehyde in PBS, then stained with a 2% crystal violetsolution. After extensive water washing to remove excess crystal violet,the number of cells that had migrated were counted in threerepresentative high power (200×) fields per insert. Data was presentedas number of cells migrating +/− standard error of measurement.

3. In Ovo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay

Ten day old embryonated chicken eggs (McIntyre Poultry; Ramona Calif.)were candled to illuminate blood vessels under the shell and an areawith a minimum of small blood vessels is identified. The CAM was droppedaway from the eggshell in this area by grinding a small hole in themineralized shell and applying pressure to the underlying inner shellmembrane. This procedure caused an air pocket to shift from the wide endof the egg to the identified area and forcing a circular region of theCAM approximately 2 cm in diameter to drop away from the shell. A windowwas cut in the egg shell and a cortisone acetate pre-treated filter disc5 mm in diameter that had been saturated in 1 μg/ml bFGF, VEGF, TNFα,IL-8 (Genzyme, Inc.; Cambridge Mass.) or saline was placed on the CAM.The window in the shell was sealed with adhesive tape and the egg wasincubated for four days.

A range of 0-25 μg in 25 μl of function blocking anti-α5β1 or a controlnon-function blocking anti-α5β1, 0-25 μM in 25 μl cyclic peptide(CRRETAWAC; SEQ ID NO: 1) or scrambled control peptide (CATAERWRC; SEQID NO: 2; Koivunen et al., J. Biol. Chem. 268:20205-20210 (1993);Koivunen et al., J. Cell Biol. 124:373380 (1994)), 0-25 μM in 25 μl ofSJ749, an inactive control small molecule or 25 μl of saline wereapplied to the growth factor saturated filter 24 hours later.Anti-fibronectin antibodies (25 μg in 25 μl) also were applied topicallyto the CAM.

Fibronectin, vitronectin and fibronectin fragments (59 pmol in a finalvolume of 25 μl) were applied to stimulated or unstimulated CAM's.Peptide or small molecule antagonists of α5β1 (at a final serumconcentration of 0-25 μM) also were injected intravenously into thechick circulation 24 hr later. CAM's were harvested on the fourth day ofstimulation. Blood vessel branch points in the 5 mm filter disk areawere counted at 30× magnification in a blinded fashion as asize-independent quantitative indicator of vascular sprouting inresponse to growth factors. Angiogenesis is characterized by thesprouting of new vessels in response to growth factors. Thus, countingof blood vessel branch points is a useful quantitative means ofobtaining an angiogenic index (Brooks et al., In “Methods in MolecularBiology” (Humana Press 1999). At least ten embryos were used pertreatment group. Each experiment was performed a minimum of three times.

Data was evaluated in terms of average number of blood vessel branchpoints per treatment group +/− standard error of measurement.Statistical analyses were performed using Student's t-test.Representative CAMS from each treatment group were photographed at 10×magnification. In some cases, CAM tissue excised from the egg was frozenin OCT (Baxter; McGraw Park Ill.) in liquid nitrogen, cut into 5 μmsections, air dried and processed for immunohistochemical analysis asdescribed in Example I, except without fixation.

4. Integrin Receptor Ligand Binding Assays

Integrin αVβ3 and α5β1 receptor purified from human placenta wereobtained from Chemicon International. Platelet integrin αIIbβ3 waspurified from platelets according to established procedures. Receptorswere coated (100 μl/well) on Costar (3590) high capacity binding platesovernight at 4° C. Coating solution was discarded and plates were washedonce with blocking/binding (B/B) buffer (50 mM Tris HCl, pH 7.4, 100 mMNaCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂ and 1% BSA).

One hundred ten microliters of B/B buffer was applied for 60 min at RT.Thirty μl of biotinylated extracellular matrix protein ligand(fibronectin for integrin α5β1, vitronectin for integrin αVβ3 andfibrinogen for integrin αIIbβ3) plus 50 μl of either SJ749 in B/B bufferor B/B buffer alone were added to each well, and incubated for 25 min atRT. Plates were washed twice with B/B buffer and incubated 1 hr at RT,with anti-biotin alkaline phosphatase (100 μl/well) in B/B buffer.Finally, plates were washed twice with B/B followed by the addition of100 μl of phosphatase substrate (1.5 mg/ml). Reaction was stopped byadding 2N NaOH (25 μl/well), and the developed color was read at 405 nm.

B. RESULTS

1. Antibody Specific for the Cell Binding Domain of Fibronectin InhibitsAttachment and Migration of Cells Expressing α5β1 Integrin toFibronectin In Vitro and Inhibits Angiogenesis In Vivo in CAM's

Since fibronectin was localized to 5β1-expressing blood vessels intumors and growth factor treated tissues, the effects of fibronectin andof function blocking anti-fibronectin antibodies on angiogenesis wasevaluated.

An in vitro cell adhesion assay was used to determine, first, whether anantibody directed against the central cell binding domain peptide(anti-CBP antibody) or an antibody against an N-terminal peptide offibronectin (anti-NT antibody) of human and chicken fibronectininhibited cell adhesion to fibronectin. The anti-CBP antibodysignificantly inhibited the adhesion to fibronectin of integrin α5β1positive cells, including α5β1⁺ HT29 colon carcinoma cells, CEF's, andHUVEC's. HUVEC adhesion was blocked 70+/−3% by the anti-CBP antibody. Incontrast, the anti-NT antibody was ineffective in blocking cell adhesionto fibronectin. These results demonstrate that the CBP domain offibronectin is required for adhesion of cells expressing α5β1 integrin.

Function-blocking monoclonal antibody antagonists of integrin α5β1, butnot control (non-function blocking) anti-α5β1 integrin monoclonalantibodies, selectively inhibited HT29 α5⁺ (100+/−6%), CEF(89.7+/−3.4%), and HUVEC (72+/−2.5%) adhesion to fibronectin, but didnot inhibit attachment to vitronectin; however, LM609, an anti-αVβ3specific antibody inhibited attachment of the cells to vitronectin.These results demonstrate that α5β1 binding to fibronectin is requiredfor adhesion α5β1 expressing cells to fibronectin.

As angiogenesis depends in part on endothelial cell migration andinvasion, the ability of anti-α5β1 antibodies to block HUVEC migrationalso was evaluated. Migration of HUVEC's on fibronectin wassignificantly inhibited (87+/−2%) by function blocking antibodiesdirected against integrin α5β1, whereas this antibody did not affectendothelial cell migration on other matrix proteins, including collagen.These results demonstrate that α5β1 integrin also is involved infibronectin mediated cell migration.

The roles of fibronectin and of α5β1 on angiogenesis was examined invivo was examined using the CAM assay. To assess the role of fibronectinin angiogenesis in vivo, CAM's from ten day old embryos were stimulatedwith bFGF or VEGF. Twenty-four hr later, anti-fibronectin antibodieswere directly applied to the CAM's, then, two days later, CAM's wereexcised and blood vessels were quantified by counting vessel branchpoints.

The anti-CBP antibody inhibited the growth of new blood vessels inducedby bFGF by 75+/−10% (p=0.002), whereas the anti-NT antibody had a onlyminimal effect (34+/−15% inhibition, p=0.02). The anti-CBP antibody alsoinhibited VEGF angiogenesis by 71+/−7% (p=0.02), as did the anti-NTantibody (89+/−17% inhibition, p=0.035).

In contrast to anti-fibronectin antibodies, function blocking antibodiesdirected against vitronectin had no significant effect on angiogenesis.These results indicate that the cell-binding domain of fibronectin playsa critical role in angiogenesis, and that the N-terminal domain offibronectin also may contribute to some angiogenesis.

To further demonstrate a specific functional association betweenfibronectin and angiogenesis stimulation, fibronectin and vitronectinwere directly applied to the CAM's of ten day old embryos in thepresence or absence of growth factors. In the absence of growth factoraddition, neither fibronectin nor vitronectin promoted angiogenesis.Equimolar amounts of intact human fibronectin, a 120 kD fragment offibronectin with the RGD containing cell binding domain or a 40 kDC-terminal chymotryptic fibronectin fragment which lacks the RGDcontaining cell-binding domain (Chemicon; Temecula Calif.) were appliedto bFGF stimulated CAM's and angiogenesis was examined.

Intact fibronectin enhanced growth factor stimulated angiogenesis atleast 46+/−11% (p=0.04). The 120 kD cell binding fragment of fibronectinalso significantly enhanced angiogenesis (65+/−20%; p=0.05); incontrast, the 40 kD fragment of fibronectin had no significant effect.Furthermore, anti-α5β1 integrin antibodies reversed this process,demonstrating that fibronectin-enhanced angiogenesis was dependent onintegrin α5β1 activity (see below). Application of vitronectin to bFGFstimulated CAM's had no effect on vessel number. Addition of fibronectinor vitronectin to VEGF stimulated CAM's also did not potentiate theangiogenic effect of VEGF. These results demonstrate that fibronectinand the endothelial cell integrin α5β1 have functional roles in growthfactor-induced angiogenesis.

The ability of anti-α5β1 antibodies to impact growth factor-inducedangiogenesis on the chick CAM also was examined. Twenty-four hr afterstimulating angiogenesis with bFGF, anti-α5β1 antibodies were directlyapplied to the growth factor saturated filter disk or were injectedintravenously into the embryonic circulation. The antibody antagonistsof integrin α5β1 blocked bFGF-induced angiogenesis on the CAM by atleast 88+/−6% (p=0.01), whereas control non-function blocking anti-α5β1antibodies had no significant effect. Applications of function-blockingor control anti-α5β1 antibodies to unstimulated CAM's had no effect onthe number or integrity of blood vessels present within the applicationarea. Similarly, anti-αVβ3 antibody also blocked angiogenesis induced bybFGF by 65+/−10% (p=0.008).

Distinct growth factors can induce selective pathways of angiogenesisthat activate or utilize distinct integrins. For example, integrin αVβ3participates in the bFGF and TNFα pathways of angiogenesis, while αVβ5participates in the VEGF and TGFα pathways. Accordingly, the role ofother integrins in growth factor induced angiogenesis was examinedfurther.

When angiogenesis was stimulated with TNFα or IL-8, anti-α5β1 antibodiesblocked angiogenesis by an average of 70.4+/−12% (p=0.04) and 85+/−4.8%(p<0.0001), respectively, and, in some experiments anti-α5β1 antibodiesinhibited TNFα and IL-8 angiogenesis by up to 99+/−5% (p=0.005).Similarly, antibody antagonists of integrin αVβ3 blocked TNFα and IL-8angiogenesis by 93.6+/−6.2% (p=0.004) and 77+/−5.2% (p=0.0001),respectively. However, when angiogenesis was induced with VEGF, antibodyantagonists of integrin α5β1 failed to block angiogenesis, whereasanti-αVβ5 antibody blocked VEGF-induced angiogenesis by 99+/−0.1%(p=0.004). When anti-α5β1 integrin and anti-αVβ3 integrin antibodieswere applied in combination to bFGF stimulated CAM's, no additive orsynergistic inhibitory effects were observed, suggesting that theseintegrins participate in the same angiogenic pathway.

These results demonstrate that an interaction of α5β1 integrin with thecell-binding domain of fibronectin is involved in growth factor-inducedangiogenesis in vivo, and that an anti-α5β1 antibody can interfere withsuch angiogenesis. The results also indicate that integrin α5β1regulates the same pathway of angiogenesis as does αVβ3 and that thispathway is distinct from that regulated by αVβ5.

2. Peptide and Nonpeptide Small Organic Molecule α5β1 AntagonistsInhibit Attachment and Migration of Cells Expressing α5β1 Integrin toFibronectin In Vitro and Inhibit Angiogenesis In Vivo in CAM's.

The ability of peptide and nonpeptide small organic molecule antagonistsof α5β1 integrin to interfere with cell interactions with fibronectinand with growth factor induced angiogenesis also was examined.

Non-antibody antagonists of integrin A51 potently inhibited cellattachment to fibronectin. The selective cyclic peptide antagonist ofintegrin α5β1, CRRETAWAC (SEQ ID NO: 1), significantly inhibitedadhesion of α5⁺ HT29 colon carcinoma cells, CEF's and HUVEC's tofibronectin, but not to vitronectin, whereas a “scrambled” controlpeptide (CATAERWRC; SEQ ID NO: 2) had little effect on cell adhesion toeither fibronectin or vitronectin. CRRETAWAC (SEQ ID NO: 1), but not thecontrol peptide, also interfered with endothelial cell migration onfibronectin, but not on other matrix proteins such as collagen. Thecyclic peptide antagonist of α5β1 also significantly blockedbFGF-induced angiogenesis (90+/−6%; p<0.0001), whereas control peptidesdid not inhibit angiogenesis. The peptide antagonists of integrin α5β1failed to block VEGF angiogenesis.

The α5β1 selective nonpeptide small organic molecule antagonist, SJ749,blocked the adhesion of these cells to fibronectin in aconcentration-dependent manner (half maximal inhibitory concentration of0.8 μM for α5⁺ HT29 cells; FIG. 1), but was ineffective in blocking cellattachment to vitronectin or other extracellular matrix ligands. SJ749also selectively inhibited ligand binding to α5β1 and was substantiallyless effective in blocking ligand binding to αVβ3 and other integrins.The nonpeptide small organic molecule antagonist of integrin α5β1 alsowas highly effective in blocking endothelial cell migration onfibronectin, but not on other matrix proteins such as collagen. SJ749also blocked bFGF-induced angiogenesis on chick CAM's in adose-dependent manner when applied either topically or systemically(FIG. 2), whereas control nonpeptide molecules did not inhibitangiogenesis, even at the highest dose tested. Like the other α5β1antagonists, SJ749 did not block VEGF angiogenesis.

These results demonstrate that peptide and nonpeptide small organicmolecule antagonists of α5β1 significantly and selectively interferewith the function of human and chick α5β1, similarly to anti-α5β1antibodies. More specifically, systemic administration of antibody,peptide and nonpeptide small molecule antagonists inhibited growthfactor-induced angiogenesis with IC₅₀'s of approximately 5 μg, 120pmoles and 15 pmoles, respectively, per 2 ml blood volume of the chickembryos. These results also confirm that the fibronectin receptorintegrin α5β1 contributes to growth factor angiogenesis on the CAM.

EXAMPLE III α5β1 Antagonists Inhibit Growth Factor Induced Angiogenesisin Human Skin in SCID Mice

This example demonstrates that α5β1 antagonists inhibit angiogenesis inhuman skin grown in SCID mice.

Engraftment of SCID mice with human skins was performed as previouslydescribed (Brooks et al., J. Clin. Invest. 96:1815-1822 (1995)). SCIDmice were engrafted with an 8 mm×13 mm piece of human neonatal foreskin.Fresh human neonatal foreskins were obtained from the Cooperative HumanTissue Network of the National Institutes of Health and were stored inRPMI-1640 medium supplemented with 2% fetal bovine serum and 1%gentamicin.

Four weeks after engraftment, after the skin had completely healed, 50μl of growth factor depleted matrigel (Becton Dickenson; Bedford Mass.)reconstituted with 1 μg/ml basic fibroblast growth factor (bFGF), with 1μg/ml bFGF containing 25 μg/ml anti-α5β1 function blocking monoclonalantibody or with 1 μg/ml bFGF containing 25 μg/ml non-function blockinganti-α5β1 monoclonal antibody was injected intradermally in the centerof each engrafted skin. Three days later, the human skin was excisedfrom the mouse. Boundaries were easily observed since the human skin waspink and hairless; the mouse skin was covered with white fur. The humanskin was embedded in freezing medium, frozen and sectioned. Sectionswere stained for the presence of human blood vessels with anti-CD31, asdescribed in Immunohistochemical analyses of blood vessel densities.Data was presented as mean CD31 positive blood vessel numbers per 100×microscopic field, +/− standard error of measurement. Statisticalanalyses were performed using Student's t-test.

Human neonatal foreskin engrafted onto SCID mice was injectedintradermally with growth factor depleted basement membrane impregnatedwith bFGF in the presence or absence of the function-blocking andcontrol anti-α5β1 antibodies. Analysis of the human skin after threedays for the presence of human CD31 positive blood vessels. The additionof function-blocking α5β1 antibody selectively blocked angiogenesisinduced by the growth factor, and reduced the number of CD31 positiveblood vessels per high power field by 94+/−4.7% (P=0.006).

These results demonstrate that integrin α5β1 has a functional role inthe angiogenic response to growth factors of human blood vessels, andthat an antagonist of α5β1 binding can reduce or inhibit growth factorstimulated angiogenesis in human skin.

EXAMPLE IV α5β1 Antagonists Inhibit Tumor Growth

This example demonstrates that α5β1 antagonists inhibit angiogenesis inhuman tumors in a CAM model system.

The chick CAM tumor assay was performed by placing ten million tumorcells on the surface of a CAM, and culturing the cells for one week. Theresulting tumors were excised and cut into 50 mg fragments. Thesefragments were placed on additional CAM's and treated topically thefollowing day with 25 μg in 25 μl of anti-α5β1 or a control non-functionblocking anti-α5β1, or systemically by intravenous injection with afinal serum concentration of 25 μM cyclic peptides or 25 μM SJ7549 and25 μM scrambled control peptide or 25 μM inactive small molecule or 25μl of saline (blood volume of chick embryo is approximately 2 ml).Forty-eight hours later, CAM's were excised from the egg and the numberof blood vessels entering the tumors were counted (as vessel branchpoints).

Data was presented as mean blood vessel number per treatment group (+/−standard error of measurement). Each treatment group incorporated atleast ten tumors per experiment. Representative tumors were photographedat 10× magnification. Tumors were excised from the egg and tumor weightswere determined for each tumor. Data was presented as mean tumor weightper treatment group (+/− standard error of measurement). Statisticalanalyses were performed using Student's t-test.

HT29 colon carcinoma cells lacking α5β1 expression were grown on theCAM's of 10-day old embryos. These tumor cells secrete severalangiogenic growth factors, including VEGF, TGFα, TGFβ, TNFα, and IL-8(Anzano et al., Cancer Res. 49:2898-2904 (1989); Varner et. al., supra,1995; Ellis et al., J. Biol. Chem. 273:1052-1057 (1998)). Integrin α5β1negative tumor cells were used to distinguish the potential anti-tumoreffects from anti-vasculature effects of integrin α5β1 antagonists.

Treatment with function-blocking, but not control, antibodiessignificant reduced (70+/−10%, p=0.02) the number of tumor-associatedblood vessels. No significant morphological or quantitative differencewas observed between saline and control antibody treated tumors or theirassociated blood vessels. Furthermore, treatment with function-blockinganti-α5β1 antibodies resulted in tumor regression. Anti-α5β1 treatedtumors were 32% smaller than control treated tumors (p=0.02).

Intravenous administration of cyclic peptide inhibitors of integrin α5β1and nonpeptide small molecule inhibitors of integrin α5β1 also inducedtumor regression on the CAM, whereas control peptide or controlnonpeptide treated tumors continued to increase in size. Tumors treatedwith peptide and nonpeptide inhibitors were 31% and 51% smaller thancontrol treated tumors, respectively (p=0.003). Tumor cells remainedintegrin α5β1 negative throughout the course of the experiment,indicating that the anti-tumor effects were based on the targeting ofthe tumor associated blood vessels.

The effect of α5β1 antagonists on tumor angiogenesis in α5β1⁺ Hep 3squamous carcinoma cells also was examined. Treatment of the tumors withfunction-blocking anti-α5β1 resulted in tumor regression, with thetumors being 45% smaller than control tumors (p=0.046). No significantmorphological or quantitative differences were observed between salineand control antibody treated tumors.

These results demonstrate that targeting vascular cell integrin α5β1inhibits tumor angiogenesis and tumor growth, and that antagonists ofintegrin α5β1 are potent inhibitors of tumor growth and tumor-inducedangiogenesis.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made with departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A method of reducing or inhibiting angiogenesis in a tissue,comprising contacting α5β1 integrin in the tissue with a peptide thatinterferes with specific binding of the α5β1 integrin to the ligandexpressed in the tissue, thereby reducing or inhibiting angiogenesis inthe tissue.
 2. The method of claim 1 wherein the peptide comprises theamino acid sequence CRRETAWAC (SEQ ID NO:1).
 3. The method of claim 1wherein the tissue is in an individual.
 4. The method of claim 3 whereinthe individual is a human.
 5. The method of claim 1 wherein the tissuecomprises a neoplasm.
 6. The method of claim 5 wherein the neoplasm is amalignant neoplasm.
 7. The method of claim 6 wherein the malignantneoplasm is selected from the group consisting of a sarcoma, amesothelioma, a teratocarcinoma, an astocytoma, and a glioblastoma. 8.The method of claim 6 wherein the malignant neoplasm is a metastaticmalignant neoplasm.
 9. The method of claim 6 wherein the malignantneoplasm is a carcinoma.
 10. The method of claim 9 wherein the carcinomais selected from the group consisting of a breast carcinoma, a coloncarcinoma, an ovarian carcinoma, and a pancreatic carcinoma.
 11. Themethod of claim 1 wherein the peptide is linked to a cytotoxin.
 12. Themethod of claim 11 wherein the cytotoxin is a cancer chemotherapeuticdrug.